Primary antibody against vinculin

Primary antibody against vinculin (Abcam, UK) was used at 1:100 dilution. Nuclei and F-actin were stained with DAPI and phalloidin respectively. Immunofluorescent staining was performed according to the manufacturer’s instructions and the images were taken by Olympus FluoView™ FV1000 confocal microscope using the same settings of laser power and pinhole size.

Although the implant is impermeable to the light, a visualized observation is still needed for a more intuitional purpose. During the experiment, MC3T3-E1 cells were seeded on implant surfaces in 24-well plates at a density of 1 × 10⁵ cells ml-1 and cultured for 24 hours and 3 Days. After removing the suspended cells with PBS, the attached cells were fixed with acetone and methyl alcohol solution and blocked with 5% FBS for 1 h. Implant was incubated with primary antibody against vinculin(1:100, Abcam, Cambridge, US) at 4 overnight and then incubated with Cy3-conjugated goat antirabbit secondary antibody(1:1000) at room temperature for 1 h. Lastly, nuclei were counterstained with 4′,6 diamidino-2-phenylindole (DAPI). The immunofluorescence were observed with an optical microscope (BX53, OLYMPUS, Japan).